Arthrosis (Arthrosis deformans) is a common degenerative disease of the joints. It involves damage (erosion) to the cartilage surfaces, detachment of cartilage particles, and inflammation of the synovial membrane caused by the cartilage particles. In cases of mild and moderate arthrosis, attempts have been made in recent years to use an intra-articular injection of hyaluronic acid (visco-supplementation) to improve the patients' pain status and to concurrently reduce the progression of the arthrosis.
Hyaluronic acid is a natural ingredient of the synovial fluid (joint liquid). Hyaluronic acid acts as a lubricant in the synovial fluid. It is particularly advantageous that aqueous hyaluronic acid solutions are visco-elastic. This results in very good lubricant and gliding properties.
Based on the advantageous lubricant properties, hyaluronic acid solutions have been in use for visco-supplementation for approximately the past two decades. The current state of the prior art is the use of hyaluronic acid, which is produced by fermentation and is used in the form of a sterile aqueous hyaluronic acid solution. Besides, the use of water-soluble cellulose derivatives, such as carboxymethylcellulose and methylcellulose, starch derivatives, such as hydroxyethyl starch, for visco-supplementation is also feasible on principle.
Thus far, it is common to sterilize aqueous hyaluronic acid solutions by exposure to gamma radiation. Doses of 25 kGy or more are common in this context. This sterilization is done on finally packaged hyaluronic acid solution.
However, the exposure to gamma radiation is associated with grave disadvantages. In addition to degradation of the polymer chains by means of which more or less low-molecular degradation products are produced depending on the dose of gamma radiation, side reactions leading to the discolouration of the hyaluronic acid solutions can occur as well. Another disadvantage of the use of gamma radiation is that the common gamma sources have a spherical radiation field. As a result, the incident doss can vary as a function of the position of the object to be sterilized. This results in uneven polymer degradation, which possibly is associated with inhomogeneities of the final viscosity. A reproducible final viscosity of the sterilized hyaluronic acid solutions is virtually impossible to attain. Moreover, the gamma radiation can lead to brittling of the packaging means, which usually are disposable plastic syringes.
Similar disadvantages are associated with steam sterilization of aqueous hyaluronic acid solutions, which can lead to damage to the hyaluronic acid and the plastic packaging means.
Due to the relatively high viscosity of the solutions, sterile filtration of aqueous hyaluronic acid solutions is basically not feasible or only with an inordinate effort. Moreover, sterile filtration removes microbial life forms only from a certain size. Viruses cannot be removed or inactivated by sterile filtration.
Aside from said physical methods, it is customary to use chemical compounds for sterilization of medical products.
These include formaldehyde, glutardialdehyde, o-phthaldialdehyde. The sterilization using aldehydes is disadvantageous in that these need to be removed again after the sterilization order to prevent damage during the use in humans. This precludes sterilization with aldehydes in the case of aqueous hyaluronic acid solutions in their final packages. Aldehydes cannot be removed again from hyaluronic acid solutions in their final packages.
Oxidising agents, such as hydrogen peroxide, performic acid, peracetic acid, hypochloride, and hypochloride-releasing substances, such as chloramine T 2 or trichloroisocyanuric acid, are very effective sterilization means. These agents are disadvantageous in that they cause significant oxidative degradation of the dissolved hyaluronic acid. Moreover, non-reacted residues of the oxidising agents may remain in the hyaluronic acid solution in its final packaging and may possibly have a local toxic effect.
It is known from pharmaceutical industry that aqueous protein solutions, such as, e.g., vaccines, are very sensitive to the effects of oxidising sterilization agents and various physical sterilization methods, for example sterilization with gamma radiation. For this reason, these aqueous protein solutions are subjected to sterile filtration first and then have small amounts of β-propiolactone added to inactivate viruses. β-propiolactone acylates the amino groups of the DNA/RNA or proteins of the viruses. The water that is present as solvent is capable of slowly decomposing β-propiolactone such that no active β-propiolactone is present any longer in aqueous protein solutions after just a short period of time. It is known thus far that gaseous β-propiolactone can irreversibly inactivate endospores (R. K. Hoffmann, B. Warshowsky: Beta-Propiolactone Vapor as a Disinfectant. Appl. Microbiol. 1958 September; 6(5): 358-362). Moreover, it is known that β-propiolactone inactivates endospores in non-aqueous organic monomers/monomer mixtures and pasty cements containing organic monomers (EP 2 596 812 B1).
However, aside from the vegetative forms, micro-organisms also have generative forms, such as endospores. These generative survival forms of micro-organisms are formed by gram-positive bacteria, in particular of the Bacillus and Clostridium genera, as a means of persisting during unfavourable living conditions. In their resting state, endospores have no active metabolism and possess a multi-layered spore capsule that largely protects the core of the spore from the action of chemicals and other environmental effects. This renders spores extremely resistant to the action of heat and chemicals (Borick, P. M.: Chemical sterilizers. Adv. Appl. Microbiol. 10 (1968) 291-312; Gould, G. W.: Recent advances in the understanding of resistance and dormancy in bacterial spores. J. Appl. Bacteriol. 42 (1977) 297-309; Gould, G. W.: Mechanisms of resistance and dormancy. p. 173-209. In Hurst, A. and Gould, G. W. (ed.), The bacterial spore. vol. 2 Academic Press, Inc. New York, 1983). Due to their high resistance, endospores are used as bio-indicators for validation and control of the efficacy of sterilization processes. This is based on the assumption that the inactivation of endospores is indicative of all vegetative microbial forms of life being killed. Endospores of gram-positive bacteria are classified in international resistance class III. Resistance classes I include non-spore-forming bacteria and vegetative forms of spore-forming bacteria and resistance class II includes spores that are killed within a few minutes in a flow of steam at 105° C. In accordance with DAB 2008 (Deutsches Arzneimittelbuch), all micro-organisms of resistance classes I-III must be killed or inactivated irreversibly.
The object of the invention is to develop a method for the sterilization of aqueous polysaccharide solutions. Said method is to enable a sterilization without significant degradation of the dissolved polysaccharide polymers.
Moreover, the sterilization method to be developed shall be suitable for sterilization of aqueous polysaccharide solutions stored in their final packages, including the internal walls of the packaging means that contact the aqueous polysaccharide solution. The sterilization methods to be developed shall safely inactivate microorganisms of resistance level III.
In the scope of the invention, sterility shall be understood to mean a state that is free of viable micro-organisms in accordance with EN 556-1:2001.